Phermthai T.Thongbopit S.Wataganara T.Wichitwiengrat S.Chuaynarong P.Poothong J.Auewarakul P.Mahidol University2025-10-062025-10-062025-09-26Stem Cell Research Therapy Vol.16 No.1 (2025) , 509https://repository.li.mahidol.ac.th/handle/123456789/112463BACKGROUND: Allogeneic mesenchymal stem cells (MSCs) offer significant advantages for various medical treatments. However, maintaining an allogeneic MSC bank presents challenges due to concerns about heterogeneity, which directly affects their efficacy. Additionally, issues related to cell senescence can arise even after a short period of serial passaging. Amniotic fluid mesenchymal stem cells (AF-MSCs) demonstrate greater proliferation efficiency compared to other MSCs. They can form clonal cell lines that generate a homogeneous population and can expand in long-term cultures without undergoing cellular senescence. Therefore, this study introduces a method for establishing cell lines and a banking system for AF-MSCs, providing high-quality, authentic human MSCs for clinical applications. METHODS: In this research, we isolated clonal AF-MSCs to select them for the creation of AF-MSC stock through long-term serial passaging. We developed a three-tier cell banking system, which includes an AF-MSC stock, a Master Cell Bank at passage 4, and a Working Cell Bank at passage 9. Standard characteristics were employed to verify identity, safety, and quality control assessments. RESULTS: Three out of twelve AF-MSC clones were selected to exemplify cell line establishment, the creation of a three-tier banking system, and quality control evaluations to determine their suitability for clinical-grade cell lines in medical applications. CONCLUSIONS: This study offers a comprehensive technical and translational overview of establishing an allogeneic amniotic fluid mesenchymal stem cell bank, tailored for medical applications and drug development.Biochemistry, Genetics and Molecular BiologyMedicineArticleSCOPUS10.1186/s13287-025-04654-22-s2.0-1050174163771757651241013842